This project investigates the function of the homeodomain-containing transcription repressors Engrailed (En) and Even-skipped (Eve). How does En locate its target genes within the nucleus? To do so, it appears to interact with the Hox protein cofactors Extradenticle and Homothorax. Each of these cofactors has been shown to be important for the activity of En in repressing a direct target gene in Drosophila embryos. The project will identify the mechanistic basis of these functional interactions in vivo. Studies of cooperative interactions on the DNA will be facilitated by the identification of direct binding sites in the target gene sloppy paired. In order to carry out its function, En has been shown to interact with corepressors, including Groucho (Gro). Like En, Eve has both a Gro interaction region and a Gro-independent repression domain. Why are two repression domains with different functional properties found in both of these developmental regulators? A transgene capable of completely rescuing eve null mutants has been constructed. Since Eve plays a major role in establishing cell fates during several stages of embryogenesis, and target genes have been well characterized, this transgene will be used to study functional distinctions among different classes of repression domain in vivo. What other partner proteins does En interact with to carry out its function? Several proteins have been identified that interact with specific functional domains of En. One interacts with the second class of repression domain in En, and genetic studies indicate that the two proteins have a close functional relationship in vivo. What other partner proteins does Ev interact with to carry out its function? Several proteins have been identified that interact with specific functional domains of En. One interacts with the second class of repression domain in Ev, and genetic studies indicate that the two proteins have a close functional relationship in vivo. Intriguingly, it is the cell cycle regulator cyclin E. This relationship may prove to be an important connection between regulation of transcription and the cell cycle. Analysis of the interaction will serve as a model for testing this hypothesis. This project will utilize genetic, cell culture, and biochemical means to study this and other novel interactions. For those interacting proteins that represent new genes, specific mutations will be identified and analyzed for their effects on the target genes of En, as well as on development generally. These studies will thus lead to a greater understanding of the regulatory mechanisms of homeodomain protein function in development and disease.